Improved TaqMan real-time assays for detecting hepatitis A virus

Abstract

Rapid advancement in genomics and bioinformatics in recent years holds great promise for research and development in many disciplines including public health. For the detection of pathogens, methods based on nucleic acid amplification need to be re-evaluated periodically to ensure the validity of signature primers and probes as more and more outbreak strains are sequenced and collected into databases in public domains. In this study, a previous assay designed computationally for detecting hepatitis A virus (HAV) was re-examined (Gardner et al., 2003). Alignment of 57 complete or near complete HAV genomes allowed identification of conserved sequences for developing new primers and TaqMan probes. Two sets of real-time reverse transcription PCR reagents were developed and characterized. These two assays had 10 to 1,000 fold lower detection limits than the previous assay when tested using representative human HAV genotypes IA, IB, and IIIA. The better of the two improved assays had a detection limit of 3.7×10−2 to 6.6×10−2 TCID50 or less. Possible causes for the improved detection sensitivity were identified and discussed. The improved HAV detection assays developed in this study would support better food safety surveillance initiatives and response to disease outbreaks of viral food-borne illness